title: Induction of autophagic cell death by a novel molecule is increased by hypoxia.
abstract: Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.
authors.affiliation: Department of Cellular and Molecular Pathology, IRCCS San Raffaele Pisana, Rome, Italy. marcotafani@yahoo.com
authors.forename: Marco
authors.initials: M
authors.lastname: Tafani
authors.forename: Luana
authors.initials: L
authors.lastname: Schito
authors.forename: Tahira
authors.initials: T
authors.lastname: Anwar
authors.forename: Manuela
authors.initials: M
authors.lastname: Indelicato
authors.forename: Patrizio
authors.initials: P
authors.lastname: Sale
authors.forename: Maura
authors.initials: M
authors.lastname: Di Vito
authors.forename: Emanuela
authors.initials: E
authors.lastname: Morgante
authors.forename: Rosanna
authors.initials: R
authors.lastname: Beraldi
authors.forename: Francesco
authors.initials: F
authors.lastname: Makovec
authors.forename: Ornella
authors.initials: O
authors.lastname: Letari
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Caselli
authors.forename: Corrado
authors.initials: C
authors.lastname: Spadafora
authors.forename: Bruna
authors.initials: B
authors.lastname: Pucci
authors.forename: Matteo A
authors.initials: MA
authors.lastname: Russo
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000578:Amidines / Q000737:chemistry / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D010588:Phagosomes / Q000378:metabolism / Q000648:ultrastructure; D013890:Thiourea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000578:Amidines; D000730:Androstadienes; D000894:Anti-Inflammatory Agents, Non-Steroidal; D000970:Antineoplastic Agents; C534043:CR 3294; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D042461:Vascular Endothelial Growth Factor A; D014268:Trifluoperazine; C025946:3-methyladenine; D013890:Thiourea; D000225:Adenine; D000077191:Wortmannin